Chemotaxis, which is characterized by directed movement of cells up a chemical gradient, is a key component in a multitude of biological processes, including neuronal patterning, wound healing, embryogenesis, and angiogenesis. The overall aim of this Program Project is to quantitatively study three distinct stages of chemotaxis in the social amoeba Dictyostelium discoideum using an approach that integrates novel experiments and mathematical modeling. Specifically, we propose to investigate chemotaxis by examining three distinct timescales: 1) Directional sensing: processes that occur on a time scale of 0-10 s and that are characterized by subcellular localization of several key signaling components but do not involve the reorganization of the cytoskeleton. 2) Formation of a stable leading edge and cell polarity: processes that occur on longer timescales (10-45 s) and that lead to cell regions that can clearly be identified as front, back and sides. Polarization can occur in response to a gradient, in which case it is coupled to the gradient sensing process, or can occur spontaneously. 3) Motility: processes that occur after the polarity is established and which include the actual movement of the cell, communication between cells at large distances and the coordinated response of large groups of cells (1-8 minutes). Our approach will rely heavily on the use of microfluidic devices which will provide us with precise control over the chemoattractant stimulus. The goal of our research is to better understand chemotaxis of eukaryotic cells. Advances in this field will benefit diagnosis and treatment of medical problems involving cell migration.